It is well documented that aggregation of high affinity IgE receptors (FceRI) on mast cells mediates allergic and hypersensitivity diseases. Not surprisingly, this receptor has been the subject of intense research investigation. Mast cells also express cell surface G protein coupled receptors (GPCRs) for a number of inflammatory ligands such as complement components C3a, C5a, neuropeptides, and chemokines, which modulate allergic and inflammatory diseases. Surprisingly, however, regulation of GPCR function in mast cells remains largely unexplored. This probably reflects the difficulty in isolating sufficient numbers of tissue mast cells for biochemical and functional studies. Furthermore, murine bone marrow-derived mast cells (BMMCs), which have been extensively used for studies on FceRI signaling, do not respond to many of the GPCR ligands. Our studies have focused on C3a receptor signaling in mast cells because of its demonstrated importance in allergic and inflammatory diseases. We found that although BMMCs express C3aR at low levels, they are weakly responsive to C3a. We therefore used two human mast cell lines (HMC-1 and LAD2) as well as primary human mast cell cultures derived from CD34+ cells. We found that these mast cells endogenously express C3a receptors (C3aR) and are highly responsive to the anaphylatoxin for signaling and mediator release. Basophilic leukemia RBL-2H3 cells express FceRI but are unresponsive to C3a. Accordingly, we found that RBL-2H3 cells stably expressing human C3aR respond to C3a for mediator release via signaling pathways that appears to be identical to that in human mast cells. Our preliminary studies revealed new roles of adapter molecules on the regulation of C3aR signaling in mast cells, which are G protein independent. C3aR is unique among GPCRs expressed in mast cells in that its carboxyl terminus possesses a class I PSD-95/Dlg/Zo1 (PDZ) motif but its role in receptor regulation is unknown. Using a newly developed proteomic array containing most of the known class I PDZ domains, we have shown that only a few of these domains associate with carboxyl terminus of C3aR (Ct-C3aR). Based on our preliminary studies, we hypothesize that interaction of C3aR with PDZ domain proteins provides a signaling platform for C3a-induced mast cell activation. In aim #1, we will delineate the role of PDZ domain adapter proteins on the regulation of C3aR function in mast cells. In aim #2, we will test the hypothesis that a second adapter molecule, b-arrestin also associates with C3aR but it modulates C3aR-PDZ domain interaction to inhibit mast cell signaling. In aim #3, we will modulate inflammation in vivo by targeting C3aR-PDZ protein interaction in mast cells. Collectively, these studies will generate significant new information on how C3aR signaling modulates mast cell function and may offer novel therapeutic approaches for the treatment of allergic and inflammatory diseases.